Calcium (Ca2+) dependent arrhythmias have been identified as a significant health problem leading to ventricular tachycardia, fibrillation and death. The exact mechanism by which these arrhythmias arise remains a critically important yet vexing problem. We hypothesize that these Ca2+ dependent arrhythmias occur through a process in which a propagating wave of elevated calcium travels through heart cells and thereby activates and entrains electrical activity. The proposals PIs (Lederer, Jafri, and Winslow) will combine state-of- the art computational modeling with novel laboratory experiments in a multi-scale approach to determine how the calcium signaling defect develops and critically test the hypothesis. This systems biology investigation will examine the molecular physiology of cardiac Ca2+ signaling at high temporal and spatial resolution under normal and pathological conditions. It will utilize the unusually powerful approach of specifically examining the molecular pathophysiology of the Ca2+ dependent arrhythmia using the molecular disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) caused by an extremely well-defined process - point mutations of critical Ca2+ regulatory proteins. We will examine how mutations in the calcium release channel (ryanodine receptor type 2, RyR2) and the Ca2+ binding protein, calsequestrin (CASQ2) contribute to Ca2+ dependent arrhythmogenesis. Mouse models of these two arrhythmias will be used to enable advanced cell biology investigations. The work will be made more general by also including an examination of Ca2+ overload arrhythmias in mouse and guinea pig. The planned investigation will encompass multiple scales: from the molecular defect, to cellular Ca2+ dysfunction to tissue arrhythmia using both mathematical modeling and biological experiments. The project will address the following four specific aims: 1) How do Ca2+sparks trigger and sustain calcium waves? 2) How do Ca2+waves propagate from cell to cell? How do Ca2+waves entrain electrical activity? 3) How do specific mutations in RyR2 and CASQ2 affect Ca2+sparks, Ca2+waves and the propagation of Ca2+waves from cell to cell? 4) How does the 3D organization of the heart affect Ca2+ entrained arrhythmogenesis? This work should provide fundamental new understanding of the heart and the role of Ca2+ in electrical dysfunction and arrhythmia and lay the foundation for new therapeutic approaches.